The Role ofEstrogen in the Mood-Lowering Effects of Acute Tryptophan Depletion in Postmenopausal Women

Laura A. Schleifer

Department of Psychology McGill University Montréal, Quebec Canada

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree ofDoctor ofPhilosophy. Submitted: August, 2001 © Laura A. Schleifer

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Abstract

Depression is a major mental health problem for women. Severallines of evidence suggest that fluctuating levels of estrogen associated with various reproductive events are related to changes in mood. It has been hypothesized that estrogen may exert its influence on mood via its effect on the serotonergic system - a system frequently implicated in the regulation of mood. The major goal of the foIlowing study was to elucidate further the role of estrogen in mood regulation. To this end, we examined the role ofestrogen in the mood-Iowering effect of Acute Tryptophan Depletion (ATD), a technique designed to cause a marked lowering of plasma and brain tryptophan, and therefore brain serotonin levels, so that the effects of decreased serotonin on mood can be studied directly. We hypothesized that 1) exogenous estrogen may protect against the mood-Iowering effects of ATD in postmenopausal women and that 2) a history of affective disturbance, particularly reproduction-related affective disturbance, would be associated with greater vulnerability to ATD as predicted by the kindling model of depression. Fifty-eight postmenopausal women were randomly assigned to treatment with estrogen (0.625 mg Premarin) or placebo in the context of a prospective, doubleblind, cross-over design. During the final two weeks of the 12-week treatment phase, aIl participants completed one ATD test session and one nutritionally balanced amino aeid control session. We found that: 1) treatment with exogenous estrogen signifieantly improved mood and menopausal symptoms as compared to placebo treatment, 2) ATD was associated with a significant lowering ofmood in both groups, 3) treatment with estrogen did not proteet women from ATD effeets unless they responded to Il weeks of

11

treatment with exogenous estrogen with enhanced mood, and 4) a history of reproduction-related affective disturbance was associated with more dysphorie mood in response to ATD. In conclusion, these data provide further evidence for a protective role of estrogen in mood regulation and for the role ofthe reproductive hormones in the kindling process of affective disturbance in women.

Table of Contents

i

lllJstract

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llcknowledgements

v

Intro 20 JUIL) were eliminated. Eight women withdrew from further participation because they had changed their minds, and 3 were advised against participation by their physician (see description ofmedical examination below).

Therefore, 72 women in total met aIl medical, psychiatrie and hormonal entrance criteria and were randomly assigned to one oftwo treatment groups: 36 participants were

55 assigned to treatment with exogenous estrogen for 12 weeks and 36 participants received a placebo for the same period of time. Groups were stratified based on past history of affective disturbance. A past history of affective disturbance was defined as one or more major depressive episodes, severe premenstrual dysphoria or postpartum depression as assessed by the

sem for DSM-IV and a reproductive history questionnaire (see

materials).

On the day of the psychiatrie evaluation and prior to any testing, all participants gave informed, signed consent by means of a form approved by a university hospital ethics committee (see Appendix 2) and were reassured that they could withdraw from the study at any time. Financial compensation was provided for lost work time and transportation costs in the following manner: (1) initial interview: $30, (2) study day 1: $40, (3) study day 2: $50.

S6

Materials:

1 - Test Battery:

a) Structured Clinical Interview for DSM-IV (SCID) (First, Williams, Gibbon, & Spitzer, 1997):

The SCID consists of a semi-structured interview designed to diagnose psychopathology using the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (APA, 1994). The length of the interview is influenced by the extent ofpsychopathology present, but it typically takes between1 'l2 and 2 'l2 hours.

b) Reproductive & Medical History:

This 22-item questionnaire (devised specifically for this study) is designed to assess reproductive history, including oral contraceptive use, menstrual cycle characteristics, premenstrual mood changes, number of pregnancies, pregnancy complications and postpartum affective disturbance and characteristics of the menopause (including date oflast menstrual period). There is also a short section on personal and family medical and psychiatrie history (Appendix 3).

57 c) Menopausal Index (Blatt, Wiesbader, & Kupperman, 1953) (revised by Sherwin, 1983):

This 27-item paper-and-pencil questionnaire was administered prior to and following hormone treatment to monitor the frequency and severity of menopausal symptoms on interval rating scales ranging from 0 (never) to 7 (very often) (Appendix 4).

d) Family Instrument for Genetic Studies (FIGS) (Nurnberger et al., 1994):

This semi-structured interview is designed to elicit information regarding family history ofpsychopathology. It can take up to 1 hour to complete.

e) Beek Depression Inventory (BDI) (Beek, Ward, Mendelson, Mock, & Erbaugh, 1961):

The BDI is a 21-item, individually administered multiple choice, self-report instrument designed for screening and quantifYing the severity of depressive affect. It generally takes 5-10 minutes to complete. Participants were instructed to assess their current mood, rather than their mood during the past week. Studies of the internai consistency and stability of the instrument indicate a high degree of reliability (Beek & Steer, 1987). Concurrent discriminative and construct validity have also been demonstrated for the BDI (Gallagher, 1986). There is a large body of evidence supporting the conclusion that the BDI has adequate reliability and validity for clinical and research purposes in general adult samples ofvarying ages (Gallagher, 1986).

58

f) Profile ofMood States - Bipolar Form (POMS-BI) (Lorr & McNair, 1982):

The POMS-BI is a 72-item, self-report affective inventory. It provides measures of 6 bipolar mood states each rated on a 4 point scale, where O=much unlike this and 3=much like this. The dimensions include: composed-anxious, agreeable-hostile, elateddepressed, confident-unsure, energetic-tired, and clearheaded-confused. It typically takes 5 minutes to complete.

g) Visual Analogue Mood Scale (VAMS) (Bond & Lader, 1974):

The VAMS consists of sixteen 1DO-mm horizontallines, each representing a bipolar dimension of mood state, on which the participant is instructed to place a perpendicular mark that best describes their mood state.

II- Drugs:

The active estrogen treatment consisted of oral conjugated equine estrogen 0.625 mg/day (premarin, Wyeth-Ayerst Laboratories, Montreal) for twelve weeks. This is the standard dose for treatment of postmenopausal women. The placebo treatment consisted of sugar piUs that were visually indistinguishable from the active hormonal tablets, administered once a day for twelve weeks.

59 Random assignment to Premarin 0.625 mg/day or to placebo was under the control of the ChiefPharmacist at the Allan Memorial Institute of the Royal Victoria Hospital, Montreal, Canada. Two random assignment lists were generated, one for women with a past history of affective disturbance and one for women with no history of affective disturbance. Drugs were dispensed by the pharmacists at the Allan Memorial Institute of the Royal Victoria Hospital, and the experimenter and the participants were blind to the treatment.

III - Amino Acid Mixtures:

The amino acid mixtures were prepared following the protocol of Young and associates (1985), and modified for use with female participants by decreasing the total amount ofamino acids in the T- drink from 100 g to 85.8 g to adjust for sex differences in body weight (Ellenbogen et al., 1996). The tryptophan deficient (T-) mixture consisted of 15 amino acids weighing 85.8 g which included: L-alanine 4.6 g, L-arginine 4.1 g, Lcysteine 2.3 g, glycine 2.7 g, L-histidine 2.7 g, L-isoleucine 6.7 g, L-lysine monohydrochloride 9.2 L-methionine 2.5 g, L-phenylalanine 4.8 g, L-proline 10.2 g, Lserine 5.8 g, L-threonine 5.4 g, L-tyrosine 5.8 g, and L-valine 7.4 g. The balanced amino acid mixture (B) contained the same amino acids plus 1.9-g L-tryptophan. Because of the unpleasant taste of methionine, cysteine and arginine, these amino acids were encapsulated and administered separately. The amino acid mixture was prepared within a few minutes of oral administration by mixing the powdered amino acids with 135 ml water, 45 ml chocolate syrup and 0.6 g of sodium cyclamate (an artificial sweetener). For

60 participants who dislike chocolate, an altemate mixture was available which consisted of the powdered amino acids, 180 ml of orange juice from concentrate, and 1. 1 g of sodium cyclamate.

IV- Hormone and Tryptophan Assays:

The free (non-aIbumin bound) plasma tryptophan concentration was assessed by determining the concentration of tryptophan found in an ultrafiltrate of plasma prepared at 25° C under an atmosphere containing 5% carbon dioxide in a centrifugai ultrafilter (MPS-l, Amicon Inc., Beverly, Mass.) through YMT membranes (Millipore Waters, Bedford, Mass.). Tryptophan in the ultrafiltrate, and in plasma, was measured by high performance liquid chromatography on a reverse-phase column (JiBondapak C 1S' Millipore Waters) with fluorometric detection.

Blood samples for the steroid hormones were centrifuged and the plasma stored at -20°C until analysis. Estradiol and estrone levels were determined in duplicate by radioimmunoassay with the use of antisera after extraction into ethyl ether. Charcoal was used as an absorbent.

61

Procedure: 1 - Design Overview:

A prospective, double-blind, placebo-controlled, cross-over design was employed. For participants who met ail inclusion criteria (hormonal, medical and psychiatric) assignment to treatment group (estrogen or placebo) was random and double-blind. Participants were also stratified based on previous history of depression. Ali participants completed one Acute Tryptophan Depletion (T-) test session and one nutritionally balanced amino acid (B) control session, scheduled at least one week apart, during the final 2 weeks of the 12-week treatment phase. Test sessions were counterbalanced for order and both participants and experimenter were blind to the nature of the amino acid mixture used during a given test session. On the day prior to each test session, participants were provided with pre-packaged, pre-cooked, low-protein meals or were instructed on how to prepare them themselves when that was more convenient for the participant (e.g. ifthey lived at a considerable distance from the testing site) (see Appendix 5). These meals, required to be eaten for breakfast, lunch and dinner on the day before the T- and B test sessions, provided 160 mg tryptophan/24 h, 22.6 g protein/24

h, and 2212 kcaV24 h and were identical to those used in previous studies (Benkelfat et al., 1994; Ellenbogen et al., 1996). A schematic diagram of the experimental design appears in Figure 2 at the end of the section.

62

11- Psychiatrie Evaluation:

Following the telephone screen, wornen who agreed to schedule an appointrnent for a psychiatrie interview (N=126) were instructed to begin an overnight fast on midnight of the night prior to their scheduled appointrnent. Prior to the initial interview, 20 ml of blood was obtained via venipuncture by a registered nurse or blood technician to determine levels ofLH, FSH, estradiol, estrone, and free (non-albumin bound) and total tryptophan.

The initial interview and baseline evaluation included the Structured Clinical Interview for DSM-IV (non-patient version), the BDI and the Reproductive and Medical History Questionnaire. Family history of psychiatrie disturbance was evaluated using the Family interview for Genetic Studies. Baseline rnood state was also evaluated, for cornparison purposes, using the rneasures that were ernployed during the testing phase. These included the POMS-BI and the Menopausal Index.

In total, the baseline test session generally took approxirnately 3-4 hours but, in sorne cases, it took as long as 5-6 hours depending on the amount of psychopathology reported by the participant.

Based on these results, participants who were deerned suitable for participation in the study (N=75) were provided with a letter for their gynecologist briefly explaining the nature of the study (see Appendix 6) and a form for their gynecologist to sign indicating

63 that the participant had no contraindications to participating in the study. Medical exclusion criteria included an estrogen-dependent cancer, breast cancer in a first-degree relative or uterine bleeding of unknown origin. The medical examiner confirmed that potential participants did not have any serious acute or chronic illnesses (e.g. diabetes or coronary heart disease (see Appendix 7). If the participant had been examined by her own gynecologist within the previous six months, the written confirmation from him/her that no contraindications to estrogen therapy existed for the participant was obtained. Women who did not have their own gynecologist were provided with a referral. Three participants were denied permission by their physicians to participate, leaving the total number ofwomen entering the treatment phase of the study at N=72. During the 12-week course of hormone treatment and prior to the Acute Tryptophan Depletion (ATD) test days, eight women withdrew (four in the placebo group and four in the estrogen group see Results section for details) leaving a total number of 64 participants. Sixty-four women entered the ATD testing phase of the study, and three withdrew (two estrogen and one placebo) between the first and second test day (two following the T- day and one following the B day), leaving a total of61 subjects who completed the entire protocol.

III - Tryptophan Depletion Test Sessions:

Following eleven weeks of treatment with either estrogen or placebo, aIl participants were scheduled for two test sessions (one week apart). On each of the test days, participants ingested one of the two amino acid mixtures (either T- or B) which were presented in a double-blind, counterbalanced fashion.

64

On the day prior to the test sessions, participants consumed only the low-protein diet provided for them and they began an overnight fast at midnight. Appointments were scheduled between 9-10 a.m. the next morning, at which time mood state was evaluated (using the POMS-BI, the VAMS, the BDI and the menopausal index) and 20 ml ofblood was obtained via venipuncture to determine plasma levels of estradiol, estrone, and free and total tryptophan. Participants were then provided with either a mixture of amino acids devoid oftryptophan (T-) or a nutritionally balanced (B) control mixture. For the next five hours, participants remained alone in a comfortable room specifically designated for ATD studies. Affectively neutral videotapes and reading material were available. Participants were asked not to sleep, and were monitored frequently. Another sample ofblood was obtained five hours following ingestion of the drink and mood state was then re-evaluated. Following testing, participants were given a tryptophan supplement and a high-protein snack to restore their tryptophan levels. The tryptophan supplement that was used is available by prescription in Canada and has not been associated with any cases of eosinophilia myalgia syndrome. Participants were supervised for up to one hour following the test session, and phone contact was maintained for up to 24 hours, ifnecessary. Ifparticipants had any questions or concerns, they were encouraged to contact the investigators.

65

Data Analysis:

Differences between the two groups with respect to demographic and baseline variables were analyzed with two-tailed, independent samples t-tests (with equal variances assumed). AlI 61 subjects were used in these analyses except where indicated.

Changes in estrone and estradiol values, hot flushes, cold sweats, and mood over the course of hormone treatment (from pre-treatment to Il weeks post-treatment) were evaluated using two-way mixed ANOVAs. Hormone Status (estrogen vs. placebo) was the between group factor, and Day (screening vs. Test day-1 (lI weeks post-treatment» was the within-subject, repeated measure.

ATD data were converted to change scores to control for intra-subject variability, and was analyzed using a two-way mixed ANOVA. Hormone Status (estrogen vs. placebo) was the between group factor, and Drink (T- vs. B) was the within-subject, repeated measure. Planned comparisons were performed for statistically significant factor effects or factor interactions.

Power Analysis:

Controlling for Type 1 error at an alpha level of .05 and power at .80, a sample size of 36 per group allowed for the detection of a difference of 0.35 standard deviations between standardized means (Cohen, 1988).

66

Ethical Issues:

I-Estrogen Replacement Therapy:

While there are several indications for estrogen replacement therapy in postmenopausal women (e.g. treatment ofhot flushes, atrophy of tissues of the reproductive tract, prevention of osteoporosis and protection against heart disease in atrisk populations), ERT is not universally prescribed and often depends on the preference of the individual patient. Assignment to the placebo group for 12 weeks, then, did not constitute a failure to provide suitable treatment for this short period of time.

For menopausal woman with an intact uterus, a progestin is usually added to the estrogen replacement regimen in order to protect against endometrial hyperplasia that could occur with treatment with estrogen alone. However, the administration of estrogen-alone for 3 months does not cause endometrial hyperplasia and quarterly administration of a progestin may, in fact, be preferable to monthly administration (Ettinger et al., 1994). Following completion of the study, medroxyprogesterone acetate (Provera) was added to the regimen ofwomen who had been in the estrogen group and who elected to continue with hormone replacement therapy (prescribed by their individual physicians). The women in the placebo group were offered active treatment when the double-blind code was broken at the conclusion of the study.

67

II-Acute Tryptophan Depletion:

As mentioned above, ATD is a technique which is short-lived, non-toxic, and makes use of nonnal metabolic processes to experimentally alter brain function. The amino acids are mixed in the same proportions as they occur in human milk and are therefore suitable for use with human participants (Young, Ervin, Pihl, & Finn, 1989). However, sorne short-tenn negative consequences of ingestion of the amino acids sometimes occur. These include: (1) feelings ofnausea or "bloating", which usually subside in one to three hours, (2) lowered or heightened energy levels (3) drowsiness or tiredness, or heightened alertness, and (4) a possible change in mood. When they occurred, these etfects were short-lasting and were usuaUy completely reversed following the balanced meal and tryptophan supplement given at the end of each study day. Participants who continued to have symptoms at the end of the day remained in the laboratory for a short time under medical observation. They were then driven home by taxi if required (N=2). They received a follow-up telephone calI that evening and on the subsequent day to ensure that the symptoms(s) had remitted.

68

ITelephone Screenin~ (initial screening and explanation of study, N=669)

!psychiatrie IntervieW! (face to face psychiatrie evaluation, baseline blood and mood testing, N=126)

\Medical Examinationl (approval for participation, N=72)

~

+

(*random assignment

~reatment group)

\Estrogenl

rlacebol

Treatment Phase

(Treatment week Il)

Test Phase: Administration ofT- and B AminoAcid Drinks (one drink per week in counterbalanced order)

[est Day 11

[est Day 11

1

1

(Treatment week 12)

[est Day 21

Figure 2: Experimental Design

[est Day 21

69

RESULTS

Seventy-two women entered the hormone treatment phase of the study. During the 12-week course of hormone treatment and prior to the ATD test days, eight women withdrew (four in the placebo group and four in the estrogen group) leaving a total number of64 participants. Two women withdrew for medical reasons (arm surgery, placebo group; unusual mammogram result, estrogen group). Four women cited side effects of the hormone or placebo treatment as their reason for withdrawing (breast sensitivity, vaginal discharge) (three in the estrogen group, one in the placebo group), one participant found full time employment and was therefore unable to commit the time (placebo group), and one woman reported anxiety regarding the testing situation (estrogen group).

Sixty-four women entered the ATD testing phase of the study, and three withdrew (two estrogen and one placebo) between the first and second test day (two following the T- day and one following the B day), leaving a total of61 subjects who completed the entire protocol. Three of the 61 women were excluded from aIl data analysis. Two of these women had been in the estrogen group and had unusual hormone profiles (e.g. extreme discrepancies between the two test days that occurred one week apart which suggested erratic compliance with hormone treatment). The data from one placebotreated woman was eliminated because her mood questionnaire data displayed patterns typical of a major depressive disorder with prominent moming symptoms, suggesting that she had become clinically depressed.

70

Therefore, the total number of participants in the hormone treatment phase analysis was 61 and the total number of participants in the ATD test phase was 58 (61 women who completed the total protocol minus the three women excluded from the analyses).

1)

Persona) and Demographie Data:

a) Age: The ages ofthe 61 participants in the study ranged from 44.9 to 63 years with a mean of52.88 years (+1- .49). Age did not differ between the Estrogen and Placebo groups (t (59)=.327, p=.745) (Table 1).

b) Weight: Weight data were not available for four participants, so mean weight was calculated based on data from the remaining 57 women. Mean weight of these participants was 153.88 (+1-3.9) pounds and did not differ between groups (t (55)=-.284, p=.778) (Table 1).

c) Menopausal History:

Of the 61 participants, 50 (82%) had undergone spontaneous menopause (i.e. nonsurgical), 2 (3%) had had hysterectomies with both ovaries left intact and therefore also

71 underwent natural menopause, 4 (7%) had had hysterectomy with unilateral oophorectomy, and 5 (8%) had had hysterectomy with bilateral oophorectomy, thus rendering them surgically menopausal (2 women from the estrogen group and 3 women from the placebo group).

Age at menopause and duration of menopause (as defined as months since last menstruation) were ascertained based on participant' s retrospective reports of their last menstrual cycle. Women who had undergone hysterectomy-only or hysterectomy with unilateral oophorectomy were excluded from this analysis. In total, 55 women were included in this analysis. The average age ofmenopause for aIl women was 49.4 (+1-.51) years, with a range of39.7 to 56 years. The two treatment groups did not differ with respect to age at menopause (t(53)=1.008, p=.318). Surgically menopausal women (n=5) had significantly earlier onset ofmenopause than naturally menopausal women (mean age=45.5 (+/-.1.93)) (t(53)=-2.517, p=.015). The average age ofmenopause excluding those who had undergone surgical menopause was 49.78 (+1-..50) years.

Mean duration of menopause (as defined by number of months since last menstruation) was 43.8 months (+1- 6.3), while median number ofmonths since last menstruation was 24 months. There were no significant differences between treatment groups with respect to number ofmonths since last menstruation (t(53)=-0.51, p=.959) (Table 1).

72 :~~~t~~ttt:~;:~:~:j:j:~:~t:~:jjj~~~~~mœill~il.I_~j~."Mi.diij~.lMiiB.Ulgiin:~:lttttttl~:t::::tt::j

Entire Sample Estrogen Group Placebo Group

Age (years)

Age at Menopause (years)

Months since Menstruation

Weight (lbs.)

52.9 (+/-.49) (n=61) 53.0 (+/-.55) (n=36) 52.7 (+/-.89) (n=25)

49.4 (+/-.51) (n=55) 49.8 (+/-.68) (n=31) 48.8 (+/-.77) (n=24)

43.8 (+/-.6.3) (n=55) 43.6 (+/-9.3) (n=31) 44.2 (+/-8.3) (n=24)

153.88 (+/-3.9) (n=61) 153.00 (+/-5.2) (n=36) 155.22 (+/.5.7) (n=25)

d) Level ofEducation:

Overall, the median classification of education level was a 2-year college degree and did not differ between groups (x 2

= 4.958, df=6; P =

.549). However, these degrees

were largely secretarial business school and hospital-based nurses' training that were popular 30 years ago and may be below the educational requirements for similar job classifications today. Alllevels of education were represented with the exception of individuals in the lowest classification ( b, P < .001

Menopausal Index Pre a

Menopausal Index Post 0

40.72 (+/- 3.65)

27.31 (+/- 3.25)

39.68 (+/- 5.65)

31.2 (+/- 4.06)

85

3) Trvptophao Levels:

a) Pretreatment Baseline Tryptophan Levels:

On the morning ofpre-treatment baseline (prior to any experimental intervention) there were no significant differences between the two groups in terms of total plasma tryptophan (estrogen group mean = 10.04 (+/-.19) J.1g/ml, placebo group mean = 9.7 (+/.20) J.1g/ml) (t(57)=1.231, p=.223» or free plasma tryptophan (estrogen group mean = 1.48 (+/-.004) J.1g/ml, placebo group mean = 1.58 (+/-.007) J.1g/ml) (t(55)=-1.249, p=.217» (table 7).

Total

Free

Estrogeo Group (0=36)

10.04 (+/- .19)

1.48 (+/- .004)

Placebo Group (0=25)

9.7 (+/- .2)

1.58 (+/- .007)

b) Post Hormone Treatment ATD Test Day Baseline Values:

On ATD test days, prior to the participants' ingestion of the amino acid drinks, there were no significant differences between the two groups in terms of total or free plasma tryptophan (T- day total plasma tryptophan: (t (56)=1.382, p=.173 ), T- day free plasma tryptophan: (t(55)=-.520, p=.605), B day total plasma tryptophan: (t(56)=.763, p=.448), B day free plasma tryptophan: (t(56)=.121, p=.904» (table 8).

86

EstrogeoGroup (0=36) Placebo Group (0=25)

T- Day Total

B Day Total

T- Day Free

B Day Free

9.76 (+/- .19)

9.91 (+/- .2)

1.56 (+/- .004)

1.64 (+/- .004)

9.33 (+/- .25)

9.68 (+/- .2)

1.61 (+/- .008)

1.63 (+/- .008)

c) Effect ofAmino Acid Drinks on Free and Total Tryptophan Levels:

Ingestion of the tryptophan defieient amino drink (T-) resulted in a signifieant reduetion in total plasma tryptophan levels in both treatment groups, while ingestion of the balaneed amino aeid drink signifieantly inereased total plasma tryptophan levels (figure 6). A two-way mixed ANOVA (with Drink and Test Time as the within group factors and Hormone Status as the between group factor) revealed a significant main effect of Drink (F(l, 56)=457.603), p8red to a plaœbo-treated group (n=24). Repeated Measures ANOVA revealed a significant effect of CÀly (p=.044) andTreatment (p=.006). Tü